Introduction

Tomato (Solanum lycopersicum L.) is the second most widely cultivated vegetable crop worldwide after potato (Food and Agriculture Organization of the United Nations [FAOSTAT], 2023). Globally, approximately 180 million tons of tomatoes are produced annually from about 5 million hectares of cultivated land (FAOSTAT, 2021). However, drought stress -exacerbated by climate change- severely reduces tomato yield and fruit quality, with potential losses ranging from 40 to 60 %, depending on genotype, developmental stage, and duration and intensity of water deficit (Grozeva et al., 2024). Drought has become one of the most critical abiotic stresses affecting global food security, as it limits crop productivity and contributes to soil degradation, desertification, and ecosystem imbalance (Chiappero et al., 2019; Chourasiya et al., 2018).

Drought disrupts several biochemical and physiological processes, which prevent plant growth, including membrane permeability, photosynthetic activity, redox homeostasis, secondary metabolism, pigmentation, turgor pressure, and relative water content (Haghpanah et al., 2024; Talbi et al., 2020). These changes are accompanied by a decline in photosynthetic rate and disruptions in carbohydrate metabolism, which together reduce biomass accumulation and overall plant vigor (Macedo et al., 2019; Wang et al., 2019).

Plants exhibit a range of adaptive responses to drought, including reducing leaf area and stomatal conductance, to limit transpiration and enhance water use efficiency (Zhu, 2016). Nevertheless, prolonged or severe stress can result in oxidative damage, disruption of cellular homeostasis, and premature leaf senescence. The magnitude of these responses largely depends on stress intensity and duration, cultivar genetics, and the plant’s developmental stage (Silva et al., 2019). Among the strategies to mitigate the adverse effects of drought, silicon (Si) application has received growing attention. Although Si is the second most abundant element in the Earth’s crust after oxygen, it is mostly present in polymerized forms that are unavailable for plant uptake (Debona et al., 2017).

Plants absorb Si as H₄SiO₄ at pH values below 9 through the root epidermis (Tubaña & Heckman, 2015), after which it is transported via the xylem to aerial tissues (Debona et al., 2017). While Si is not considered an essential nutrient, increasing evidence demonstrates its beneficial effects on plant performance under biotic and abiotic stress conditions (Chen et al., 2018). In particular, Si supplementation has been shown to enhance chlorophyll content, photosynthetic efficiency, nutrient uptake, and antioxidant defense, while reducing transpiration and oxidative damage under water deficit (Coskun et al., 2016; Haghpanah et al., 2024).

Given these properties, the present study aimed to investigate the effects of silicon supplementation and drought stress on the physiological and biochemical responses of tomato plants.

Material and methods

Experimental design

The experiment was conducted in 2021 at the laboratories and greenhouse facilities of the Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari, Northern Iran. A completely randomized factorial design was established with three replications. Treatments consisted of four Si concentrations in the Hoagland nutrient solution (0, 0.75, 1.5, and 3 mM) and three drought stress levels based on field capacity (FC): control (80 % FC), moderate stress (60 % FC), and severe stress (40 % FC).

Plant material and growth conditions

Tomato seeds (Solanum lycopersicum cv. CH Falate) were sown in 5-L pots filled with a 1:1 mixture of loam soil and decomposed manure. The experiment was conducted in a greenhouse under controlled conditions: 20-22 °C (day/night), relative humidity of 50-60 %, 12-h photoperiod, and light intensity of 8.5 klux. Plants were irrigated every three days with 150 mL of water and fertilized weekly with the Hoagland nutrient solution (Hoagland & Snyder, 1933).

Drought stress application

FC was determined by saturating the substrate, allowing free drainage for 48 h, and recording the pot weight. Water stress treatments were imposed during the flowering stage (50-70 days after seedling emergence) at three levels (80, 60, and 40 % FC). Target pot weights for each treatment were calculated gravimetrically. Pots were weighed daily using a precision balance (± 0.5 g), and water was added to maintain the target weight. Silicon treatments (0, 0.75, 1.5, and 3 mM) were applied via irrigation 28 days before drought induction following the method of Diogo and Wydra (2007).

Relative water content

To calculate leaf relative water content (RWC) (Ritchie et al., 1990), four leaves from each plot (with the same leaf weight in all treatments) were evaluated, and fresh tissue mass (W _f ) was measured using an analytical balance. Samples were then rehydrated in distilled water for 24 h to determine turgor mass (W _t ), removing excess water with paper towels. Dry mass (W _d ) was obtained by placing the leaves in an oven at 70 °C for 48 h. RWC values were calculated using the following equation:

Enzyme activity and protein quantification

Frozen leaves (200 mg∙plant^-1) were pulverized using a mortar previously frozen with liquid nitrogen. The powdered samples were mixed with 1.5 mL of potassium phosphate buffer (pH 6.8) containing 0.5 mM EDTA and centrifuged at 12 000 rpm for 20 min at 4 °C. The supernatant was used for enzyme activity assays (Haghpanah et al., 2024). Protein concentration was determined using the Bradford (1976) method, with bovine serum albumin as a standard (R² = 0.98; y = 0.0103x + 0.0028). Absorbance was recorded at 595 nm using a UV-Vis spectrophotometer (T92⁺Double® Beam, PG Instruments, England).

Proline content

The proline content in the leaves was obtained from the methodology of Bates et al. (1973). The results were expressed in micrograms of proline per gram of fresh weight (µg∙g^-1 FW).

Malondialdehyde content

Malondialdehyde (MDA) content was measured according to Ohkawa et al. (1997). Absorbance was recorded at 450, 532, and 600 nm, and MDA concentration was calculated as:

Hydrogen peroxide content

Hydrogen peroxide (H₂O₂) concentration was determined following the method described by Alexieva et al. (2001), which involved reacting the extract with potassium iodide (KI). The results were expressed in micromoles of H₂O₂ per gram of fresh weight (µmol∙g^-1 FW).

Superoxide dismutase activity

Superoxide dismutase (SOD; EC 1.15.1.11) activity was determined according to Patykowski and Urbanek (2003) with slight modifications. The 1.5 mL reaction mixture contained 100 µL enzyme extract, 1.5 mM EDTA, 10 mM methionine, 75 µM NBT, and 50 mM potassium phosphate buffer (pH 7.4). Two milligrams of riboflavin were added, and the mixture was exposed to a 15 W fluorescent lamp for 10 min. One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of NBT photoreduction. Absorbance was measured at 560 nm, and enzyme activity was expressed as U·mg^-1 protein (Haghpanah et al., 2024).

Phenylalanine ammonia-lyase activity

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity was determined following Lisker et al. (1983) with some modifications. A total of 100 µL of enzyme extract was combined with 500 µL of 50 mM Tris-HCl buffer (pH 7) and 60 µL of 10 mM phenylalanine. The mixture was incubated at 37 °C for 1 h with constant shaking at 300 rpm; subsequently, 1 mL of toluene and 250 µL of 4 N HCl were added. The samples were vortexed for 30 s and centrifuged at 1 000 rpm for 2 min. The absorbance of the supernatant was measured at 290 nm. The results were expressed as U·mg^-1 protein (Haghpanah et al., 2025).

Catalase activity

Catalase (CAT; EC 1.11.1.6) activity was determined according to Aebi (1974). The reaction mixture (3 mL) contained 500 µL enzyme extract, 50 mM H₂O₂, and 100 mM potassium phosphate buffer (pH 7.0). The decomposition of H₂O₂ was monitored at 240 nm, and enzymatic activity was expressed as U∙mg^-1 protein∙min^-1.

Peroxidase activity

Peroxidase (POD; EC 1.11.1.7) activity was assayed using the methodology of Nickel and Cunningham (1969). The reaction mixture (2.8 mL) contained 9 mM guaiacol, 2 mM H₂O₂, and 50 mM potassium phosphate buffer (pH 6.5). The reaction was initiated with 50 µL of enzyme extract, and the increase in absorbance at 470 nm was monitored for 3 min. Activity was expressed as U∙mg^-1 protein∙min^-1.

Statistical analysis

Data were analyzed using analysis of variance and comparison of means using the protected Fisher's test (LSD, p < 0.01). Analyses were performed using SAS ver. 9.1 software (SAS Institute, 2001).

Results and discussion

Analysis of variance revealed that both Si concentration and drought severity significantly (p < 0.01) affected fresh and dry biomass (Table 1).

Table 1. Variance analysis of the effect of water stress and application of silicon on tomato plants.

Overall, increasing drought intensity significantly reduced plant biomass regardless of Si level. Plants under severe drought stress (40 % FC) and 3 mM Si exhibited the lowest fresh weight (2.1 g), representing a 183 % reduction compared to the control (80 % FC). In contrast, the highest fresh weight (5.59 g) was observed in plants irrigated with the nutrient solution containing 1.5 mM Si and without drought stress (Figure 1), suggesting this concentration as optimal for biomass yield in tomato. These findings are consistent with previous studies reporting enhanced biomass production in tomatoes (Turan et al., 2023) and other vegetable crops (Macedo et al., 2019) following Si supplementation.

Similarly, the highest dry weight was observed in the treatments without drought stress with the application of 0, 1.5, and 3 mM Si, while the lowest dry weight (0.19 g) was recorded in the treatment with severe drought and 1.5 mM Si (Figure 2). The positive effects of Si can be attributed to the deposition of silica in cell walls (Coskun et al., 2023), improved photosynthetic efficiency (Dou et al., 2023), and enhanced antioxidant enzyme activity (Hasanuzzaman et al., 2022).

The results obtained highlight the importance of optimizing Si concentrations for achieving maximum efficiency under drought stress conditions. The biomass enhancement observed supports previous evidence that Si contributes to maintaining cellular turgor and structural integrity under water deficit (Etesami & Jeong, 2018). Furthermore, comparative studies show that the optimal Si concentration varies among species and environmental conditions, with 1.5 mM in tomato versus 2 mM in cucumber (Kim et al., 2023).

Relative water content

RWC decreased significantly (p < 0.01) with increasing drought intensity at all Si concentrations (0, 0.75, 1.5, and 3 mM). Under severe stress, plants treated with 1.5 mM Si exhibited the lowest RWC (30 %), whereas the control treatment (80 % FC) maintained the highest value (100 %) (Figure 3). Despite the overall decrease in RWC with drought, Si application at 0.75 and 3 mM significantly mitigated water loss under severe stress conditions compared to the treatment without Si supplementation.

These findings are consistent with those of Yahyaabadi and Asgharipour (2015), who observed a decline in RWC with increasing drought in fennel (Foeniculum vulgare Mill.). Similar trends have been reported in cereals such as Triticum aestivum, Aegilops tauschii, Aegilops crassa, and Aegilops cylindrica (Pour-Aboughadareh et al., 2019), and in wheat under moderate and severe water stress (Zhao et al., 2020). Turan et al. (2023) showed that when tomatoes were stressed by drought, their shoot fresh weight decreased by 43 %. The observed improvements in RWC under Si supplementation are attributed to silica deposition in the leaf apoplast, which reduces transpirational water loss and enhances water retention (Gonge et al., 2005). As RWC is closely linked to leaf photosynthetic capacity, its maintenance under drought indicates physiological resilience and reduced cellular dehydration (España-Boquera et al., 2010).

Proline content

Analysis of variance indicated significant effects (p < 0.01) of Si, drought stress, and their interaction on proline accumulation (Table 1). Proline content increased progressively with drought severity, with the highest concentration (3.74 µg∙g^-1 FW) observed in plants treated with 3 mM Si under severe stress, representing a 306 % increase relative to the control (80 % FC) (Figure 4).

Proline accumulation under drought stress is a well-documented adaptive response, as it contributes to stress tolerance (Stewart & Lee, 1974; Wang et al., 2015). Similar increases to those obtained in the present study were reported by Yahyaabadi and Asgharipour (2015) in fennel subjected to 40 % FC and foliar application of 10 mM Si, and by Ahsan et al. (2023) in drought-stressed gerbera. Aazami et al. (2021) investigated proline concentrations in some tomato cultivars and observed that proline accumulated significantly in plant tissues under stress conditions. Proline stabilizes cellular macromolecules, preserves enzyme function, and scavenges reactive oxygen species (ROS) (Bacha et al., 2017; Das & Roychoudhury, 2014). Si application further enhances proline and osmolyte accumulation, strengthening the plant’s stress defense capacity (Ahmad et al., 2008; Schobert & Tschesche, 1978) and reducing MDA (Song et al., 2024).

Enzyme activity and related components

The interaction between Si concentration and drought stress significantly affected H₂O₂ and MDA accumulation (p < 0.01) (Table 1). H₂O₂ levels increased with drought severity across all Si treatments, with the highest concentrations observed under severe stress (Figure 5). In contrast, MDA levels peaked under moderate stress in Si-treated plants, suggesting maximum membrane lipid peroxidation at this stage (Figure 6). Song et al. (2024) mention that Si-treated tomatoes maintain cellular integrity under drought conditions, observing a 21.5 % decrease in MDA levels compared to treatments without Si. The observed pattern indicates a non-linear oxidative response, where moderate stress induces the most significant membrane damage, while severe stress triggers compensatory antioxidant mechanisms.

Drought and Si treatments also modulated antioxidant enzyme activity. SOD activity increased slightly under severe drought, reaching its maximum at 3 mM Si (Figure 7). PAL activity followed a similar trend, showing the highest induction at 3 mM Si under severe stress (Figure 8). CAT activity decreased significantly (p < 0.01) under severe drought in the absence of Si, but was significantly improved with the application of 0.75 and 1.5 mM Si (Figure 9). POD activity increased with drought intensity, with the highest values recorded under severe stress and 3 mM Si (Figure 10). Overall, these results indicate that Si supplementation, especially at higher concentrations, reinforces the antioxidant defense system (SOD, CAT, POD) and enhances PAL activity, thereby mitigating oxidative damage during drought (Haghpanah et al., 2021). This suggests that Si plays an important role in alleviating oxidative damage associated with water deficit.

The results obtained are consistent with previous reports, which show that Si supplementation enhances SOD and CAT activity by 42 and 31 %, respectively, in drought-stressed tomatoes (Cao et al., 2015, 2017). Naz et al. (2021) observed that Si, enhanced with hydrogen sulfide (H₂S), improved SOD and CAT activities under combined drought and heat stress conditions. The coordinated increase in enzymatic antioxidants indicates the role of Si in maintaining redox homeostasis, especially during prolonged water deficit.

Habib et al. (2022) mention that Si-mediated induction of antioxidant enzymes maintains redox balance by mitigating oxidative damage and accelerating H₂O₂ degradation. The dual function of Si, allowing a slight accumulation of H₂O₂ for stress signaling while preventing cytotoxic levels, was corroborated in wheat, where nano-Si optimized ROS dynamics through phytolith-mediated H₂O₂ scavenging (Abdo et al., 2024). Compared with alternative drought mitigation agents -such as selenium nanoparticles (Ishtiaq et al., 2023), salicylic acid (Rai et al., 2024), algae-based bio-stimulants (Cerruti et al., 2024), and vanillic acid (Parvin et al., 2024)- Si offers a broader spectrum of protection, coordinating enzymatic (SOD/CAT), non-enzymatic (proline/GSH), and structural responses (cell wall silicification), as well as improved stress tolerance and increased tomato yield (Cao et al., 2017; Habib et al., 2022; Ishtiaq et al., 2023).

The results demonstrate that Si application alleviates the adverse effects of water deficit in tomato plants by improving antioxidant enzyme activity, reducing oxidative stress, and sustaining biomass accumulation. The responses varied depending on the Si concentration and the degree of stress, demonstrating the influence of numerous factors. Si emerges as a multifaceted and eco-friendly amendment capable of strengthening drought resilience in tomato plants by integrating biochemical, physiological, and structural defense mechanisms.

Conclusion

This study provides strong evidence that silicon supplementation effectively modulates the physiological and biochemical responses of tomato plants under drought stress. Under severe water deficit (40 % FC), the application of 0.75 mM Si significantly enhanced SOD activity, indicating its role in protecting cells against oxidative stress.

Although H₂O₂ accumulation increased with drought severity, Si-treated plants exhibited enhanced stress tolerance. Plants exposed to severe stress showed the lowest fresh (2.1 g) and dry (0.19 g) biomass, representing a 183 and 168 % reduction compared to the control, respectively. Beyond mitigating biomass loss, silicon contributes to sustaining plant vigor and metabolic balance under water-limited conditions. These results highlight the potential of Si as a cost-effective and environmentally sustainable amendment for improving drought resilience in tomato cultivation. Future studies should explore the underlying molecular and signaling pathways associated with Si-mediated drought tolerance and assess its optimal dosage and mode of application across different tomato genotypes and environmental scenarios to enhance both yield and fruit quality.